Human pluripotent stem cells (hESCs) hold remarkable potential for regenerative medicine, drug screens and basic research on human diseases. However, a number of technical hurdles still limit our ability to fully unravel their potential for therapeutic applications and basic research. One such hurdle is the difficulty in generating transgenic hESC lines and the use of reporter systems or gain- or loss-of-function approaches.
Currently, gene transfer into hESCs is mostly based on the use of viral vectors. These vectors can mediate transgene expression in 20-80% of human ES cells (Pfeifer, A., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99: 2140-2145; Ben-Dor, I., et al. (2006) Mol. Ther. 14: 255-267) but their cargo size is restricted to about 5 Kb and their insert is often limited to one expression cassette. Non-viral systems using the Sleeping Beauty transposon are also restricted to a cargo size of 5-6 Kb (Wilber, A., et al. (2007) Stem Cells 25: 2919-2927). These characteristics limit the use of selectable markers, inducible cassettes, insulators and large regulatory sequences often required to restrict transgene expression to specific cell types. Baculoviruses can deliver larger cargo to hESC (Zeng, J., et al. (2007) Stem Cells 25: 1055-1061) but the long-term effect of adeno-associated rep protein expression could lead to undesirable effects (McCarty, D. M., et al. (2004) Annu. Rev. Genet. 38: 819-845). In principle, plasmids and bacterial artificial chromosomes (BACs) can be used when large promoters are required, but stable integration of these vectors in the hESC genome is relatively inefficient and plasmid-borne transgenes are subject to silencing (Braam, S. R., et al. (2008) Nat. Methods 5: 389-392). In addition, BACs usually carry several intact genes in addition to the engineered locus, therefore their integration in the genome leads to gene multiplications. This may have important consequences when the supernumerary genes have regulatory functions.
Regardless of efficiency, gene transfer technologies currently available in hESCs such as viral vectors, Sleeping Beauty transposons or PhiC31 integrase (Thyagarajan, B., et al. (2008) Stem Cells 26: 119-126) mediate irreversible genome modifications. This constitutes a major barrier to the clinic because the presence of exogenous inserts in certain loci may lead to higher predisposition towards tumorigenesis or uncontrolled cellular behavior.
Most if not all gene delivery systems fall into two categories: systems that mediate irreversible gene integrations (e.g. viral vectors, PhiC31-based systems, plasmids and BACs) and systems that can be excised but leave mutations in the host genome upon excision (e.g. Cre-, Flp- or Sleeping beauty-based systems). These irreversible genetic alterations could potentially lead to higher predisposition towards uncontrolled cellular behavior and they constitute a significant barrier to many clinical applications including the use of hESCs.
The Lepidopteran transposable element piggyBac is capable of excision from the host genome without leaving trace mutations and has been shown to be operable in a wide range of host cells. The PiggyBac transposon isolated from the cabbage looper moth Trichoplusia ni (Fraser, M. J., et al. (1996) Insect. Mol. Biol. 5: 141-151) is non-viral, can carry cargo sizes of up to 14.3 Kb, and exhibits higher activity than other transposons in mammalian models (Ding, S., et al. (2005) Cell 122: 473-483; Wilson, M. H., et al. (2007) Mol. Ther. 15: 139-145; Cadiñanos, J., et al. (2007) Nucleic Acid Res. 35: e87). Importantly, PiggyBac does not leave footprint mutations upon remobilization (Wilson, M. H., et al. (2007) Mol. Ther. 15: 139-145). A mouse codon-optimized version of the PiggyBac transposase coding sequence (CDS) has also been disclosed and shown to provide increased transposition levels in murine embryonic stem cells (Cadiñanos, J., et al. (2007) Nucleic Acid Res. 35: e87).
Such a fully reversible method of gene delivery that minimizes concerns of permanent genetic alteration would be desirable in the development of clinically useful therapeutic stem cell applications. However, the wild-type piggyBac element is still limited by the size of its cargo (i.e. transposon insert size) and its transposition efficiency.